JOURNAL OF BACTERIOLOGYVol. 88, No. 1, p. 143-150 July, 1964Copyright a 1964 American Society for Microbiology

Printed in U.S.A.

INDUCTION AND MECHANISMS OF ARSENITE RESISTANCEIN PSEUDOMONAS PSEUDOMALLEI

KEI ARIMA AND MICHIKO BEPPU

Laboratory of Fermentation, Department of Agricultural Chemistry, University of Tokyo, Tokyo, Japan

Received for publication 2 March 1964

ABSTRACT

ARIMA, KEI (University of Tokyo, Tokyo,Japan), AND MICHIKo BEPPU. Induction andmechanisms of arsenite resistance in Pseudo-monas pseudomallei. J. Bacteriol. 88:143-150.1964.-Pseudomonas pseudomallei strain 54, ableto grow in the presence of 2 X 10-2 M arsenite,was isolated from soil. After a short lag period, itgrew at a normal growth rate. In the organismsgrown with 10-2 M arsenite, oxidation of a-keto-glutarate and other substrates proceeded in thepresence of the same concentration of the drug.The concentration of arsenite which was half-inhibitory to a-ketoglutarate oxidation was 1.6 X10-3 M in the sensitive bacteria and 3.3 X 10-2 Min the resistant ones. Cells capable of oxidizinga-ketoglutarate in the presence of arsenite wereinduced rapidly by contact with arsenite in grow-ing cultures; when the drug was removed from thecultures, resistance was maintained for abouttwo generations and then gradually disappeared.From the data presented, it was concluded thatresistance in this organism is a physiologicalchange and not a hereditary one. Further studieswere carried out to investigate the arsenite-resistance mechanisms. a-Ketoglutarate dehy-drogenase activity in the cell-free extracts ofthe resistant bacteria was sensitive to arsenite.An increase in the contents of this enzyme andsulfhydryl compounds, involving lipoic acid, wasnot observed in the resistant bacteria. The possi-bility of detoxication of arsenite was ruled out.Treatment of the resistant cells with cetyl-tri-methylammonium bromide made them suscepti-ble to 2 X 10-2 M arsenite, although untreatedcells were resistant to the same concentration ofthe drug. These data suggest the decreased per-meability to arsenite of the resistant bacteria as a

main mechanism of resistance.

It is a well-known concept that microorganismshave extraordinary adaptability to changes of en-vironmental conditions; an important example ofsuch ability is the phenomenon of drug resistance.Despite their great theoretical interest and clini-

cal importance, the biological mechanisms under-lying the development of bacterial resistance tovarious drugs are at present imperfectly under-stood. To study drug resistance in microorgan-isms, it may be of some advantage to use strainsresistant to enzyme inhibitors, since their struc-tures are more or less simple and their inhibitingmechanisms have been established. For the pur-pose of investigating drug resistance, we isolatedfrom soil one strain which was highly resistant toarsenite. Arsenite is a well-known specific inhibi-tor to the dithiol enzyme, i.e., pyruvate anda-ketoglutarate dehydrogenases. The organismwas named Pseudomonas pseudomallei strain 54and was examined for its mechanism of resistanceto arsenite.

Resistance to various drugs in living microor-ganisms can take place both at a populational andindividual level. For instance, inheritable ac-quisition of resistance to various antibiotics occursthrough mutation and selection (Newcombe andHawirko, 1949), whereas physiological adaptationis also found in the drug resistance of variousmicroorganisms (Shrint and Kelly, 1962; Ashidaand Nakamura, 1959).

In the present report, evidence is presented forphysiological acquisition of arsenite resistance inP. pseudomalli 54. Data are also given which sug-gest the decreased permeation of arsenite into thecell as a main mechanism of resistance in this or-ganism. A succeeding report will elucidate thispoint further.

MATERIALS AND METHODS

Organisms and media. Arsenite-resistant P.pseudomallci 54 isolated from soil was usedthroughout this study. Bouillon-peptone brothwas used for cultures of bacteria.

Procedure for isolation of drug-resistant microor-ganisms. After inoculation of a drop of soil sus-pensions into medium containing 2 X 10-2 Msodium arsenite, stationary cultivation was car-

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ARIMA AND BEPPU

ried out overnight at 30 C. When marked pro-liferation of microorganisms was observed, thecultivated media were spread, after appropriatedilution, on an agar plate of medium containingthe same concentration of the drug. The microor-ganisms thus isolated were stocked on agar slantscontaining the drug.

Conditions and measurements of growth. Theorganisms were grown overnight aerobically at30 C in bouillon-peptone broth, and samples ofthis culture were inoculated in the same mediumwith or without arsenite. Cultivation was under-taken in L-type test tubes tilting slowly at 30 C.Turbidity was measured photometrically with aKotaki nephelometer. To prepare cell-free ex-tracts, large-scale cultivation was undertaken in500-ml Sakaguchi flasks by reciprocal shaking.

Preparation of cell-free extracts. Organismsgrown in the presence or absence of 10-2 M ar-senite were harvested in the middle of exponentialgrowth. The washed cells were suspended inphosphate buffer (0.2 M, pH 7.4) and were dis-rupted by treatment in a sonic oscillator (ToyoRiko, 10 kc) for 5 min. After removal of cell debrisby centrifugation at 10,000 X g for 10 min, thesonic extracts were used as crude cell-free prep-arations.

MIanometric measurements. Oxidation of varioussubstrates by P. pseudomallei 54 was measured byWarburg manometric techniques. The cells wereharvested in the middle of the logarithmic growthphase and suspended in phosphate buffer (0.2 M,pH 7.4). Warburg vessels contained apppopriateamounts of cell suspension, 100 ,umoles of phos-phate buffer (pH 7.4), and 20,umoles of thesubstrates (total volume, 2.0 ml; gas phase, air;temperature, 30 C).

Assay of a-ketoglutarate dehydrogenase in thecell-free preparations. Reduction of nicotinamideadenine dinucleotide (NAD) was measured spec-trophotometrically at 340 m,.u with a HitachiEPU-2 spectrophotometer; the cell (light path, 1cm) contained 1 ,umole of NAD, 0.1 mg of co-enzyme A (CoA), 0.2 ,umole of cocarboxylase, 0.3,4mole of MgCl2, 10,umoles of L-cysteine, 5,molesof a-ketoglutarate, 80 Amoles of phosphate buffer(pH 7.4), and 0.2 ml of crude cell-free extract.Arsenite was added if necessary. The final volumewas adjusted to 3 ml. The reaction was startedby the final addition of a-ketoglutarate. One unitof the enzyme was defined as that amount whichcaused an initial rate of increase in optical density

at 340 m,u of 0.001 per min under the conditionsdescribed above.

Analytical methods. Total content of lipoic acidin the cells was estimated by bioassay with Strep-tococcus faecalis after the hydrolysis of the cellsaccording to the method of Wagner et al. (1956)or Stockstad et al. (1960).

Total free sulfhydryl compounds were esti-mated by the method of Saville (1958) after de-proteinization of each material.

Estimation of arsenite was carried out by io-dometry (Feigel, 1954). Protein concentrationwas measured by the micro-Kjeldahl method.

Biochemical reagents. NAD, CoA, and cocar-boxylase were purchased from Mann ResearchLaboratories, Inc., New York, N.Y.

RESULTS

Isolation of drug-resistant microorganisms fromsoil. Seven strains of bacteria able to grow on themedium containing 2 X 10-2 M arsenite could beisolated from soil. For further study, we chose onebacterial strain, 54, which could grow on mediumcontaining 10-2 M arsenite. Strain 54 can oxidizea-ketoglutarate rapidly. It was identified as be-longing to P. pseudomallei according to Bergey'sManual of Determinative Bacteriology. Taxonomicdescriptions were as follows.Rods 0.3 to 0.5 by 0.8 to 1.8 ,u. Motile by means

of polar flagella. Gram-negative.Agar colonies: Circular, convex, smooth.Agar slant: Growth moderate, butyrous,

smooth.Nutrient broth: Turbid.Gelatin stab: Liquefaction.Litmus milk: Slightly acidic.Acid formed from glucose.Starch not hydrolyzed.H2S not produced.Citrate not used as a sole carbon source.Oxidase activity test by Nadi-reaction: Nega-

tive.Indole production: Negative.Nitrite not produced from nitrate.Effect of arsenite and arsenate on growth. The in-

hibitory effect of serially increased concentrationsof arsenite on growth of P. pseudomallei 54 is il-lustrated in Fig. 1. A progressive increase in thelag period was observed with increasing concen-trations of arsenite, but the exponential growthrate and final amount of growth equalled that of

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VOL. 88, 1964 INDUCED ARSENITE RESISTANCE IN P. PSEUDOMALLEI

the uninhibited control. Complete inhibition oc-curred at a concentration of 3 X 10-2 M arsenite.To determine whether cultures that had re-

sumed growth now became resistant to arsenite,cells were prepared for inoculation as describedabove and inoculated into fresh medium contain-ing 10-2 M arsenite. Figure 2 shows that the pro-longed lag typical of adapting cultures waseliminated or shortened by preliminary growthin the presence of arsenite. This shows that thecells acquired a resistant character during growthwith arsenite.The growth of bacteria subjected to prelim-

inary growth with 10-2 M arsenite was also re-sistant to 3 X 10-2 M arsenate, although thisconcentration of arsenate was sufficient to depressthe growth rate of the unadapted organisms (Fig.3A). Conversely, organisms grown in the presenceof 10-2 M arsenate were able to grow without any

200 F

100lo

I/ 3 A10°l-~~~~~~~-l

I

0 2 4 6

Hours

FIG. 1. Effect of various concentrations of arse-

nite on growth of Pseudomonas pseudomallei 64.The numbers indicate molarity of arsenite.

HoursFIG. 2. Effect of 10-2M arsenite on growth of the

bacteria after preliminary growth in the presenceof various concentrations of the drug. The numbersindicate molarity of arsenite to which the cellsadapted during preliminary growth.

inhibitory effect when they were transferred tothe medium containing 10-2 M arsenite (Fig. 3B).

Effect of arsenite and arsenate on oxidation ofa--ketoglutarate. Oxidation of the various sub-strates by the cells which had grown in the pres-ence of 10-2 M arsenite was found to be resistantto the same concentration of arsenite (Table 1).In the case of a-ketoglutarate oxidation, whichmay be a key reaction inhibited by arsenite, oxy-gen uptake became completely resistant towardarsenite, and a slight activation was sometimesobserved. Thus, oxidation activity of a-keto-glutarate in the presence of arsenite might be usedas a measure of arsenite resistance.

Inhibition of a-ketoglutarate oxidation was

200

control( -10-2, 10-3 10-4

105

10-6iook

:2)S..6

._i

4-

50 I

0

lOt

501--o

72

2)

0 2 4 6

10 H

5

l

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ARIMA AND BEPPU

200

Hours Hours

FIG. 3. Mutual cross-resistance to arsenite andarsenate inhibitions observed with bacterial growth.(A) Effect of 3 X 10-2 M arsenate on growth of un-

adapted bacteria (A) and adapted bacteria afterpreliminary growth with 10-2 M arsenate (0).Culture without arsenate is the control (0). (B)Effect of 10-2 M arsenite on growth of the unadaptedbacteria (A) and adapted ones after preliminarygrowth with 10-2 M arsenate (0). Culture withoutarsenite is the control (-).

TABLE 1. Effect of 10-2 M arsenite on oxidation ofvarious substrates by sensitive and resistant cells

Sensitive cells Resistant cells

Substrate Qo° Qo2Qo* with Inhibi- Qo, with Inhibi-

senite senite

Pyruvate... 62.5 8 87.2 41.2 52 (12.5)ta-Ketoglu-

tarate .... 210 22 89.5 204 191 6.5Succinate ... 290 81 72 278 278 0Malate. 282 35 35 300 300 0

* Expressed as ,umoles of oxygen consumed permg of cell N per 30 min at 30 C.

t Stimulation.

measured at various concentrations of arsenitewith the organisms which were grown in the ab-sence or presence of 10-2 M arsenite. When theinhibition ratio was plotted against the concentra-tion of arsenite, the inhibition curves illustratedin Fig. 4 were obtained. The concentration ofarsenite required for 50% inhibition was about

1.6 X 10-3 M in the sensitive cells and about3.3 X 10-2 M in the resistant cells grown in thepresence of 10-2 M arsenite; i.e., the latter was

about 20 times higher than the former. Not onlya great difference in the half-inhibitory concen-

trations, but a considerable change in the shapeof these curves, is observed with resistant cells.

Cross-resistance to arsenite of the arsenate-grown organisms was also observed with oxidativeactivity of a-ketoglutarate. Oxidation of a-keto-glutarate in cells grown in the presence of 10-2 or

10-2 M arsenate became resistant to arsenite at a

concentration of 10-2 M (Table 2).Appearance and disappearance of resistance to

100 ------_ _ _

501/

0 0.01 0.02 0.03 0.04

Arsenite concentration (M)

FIG. 4. Inhibition by arsenite of the oxidationof a-ketoglutarate by the cells grown in the absence(0) and in the presence (0) of 10-2 M arsenite.

TABLE 2. Effect of 10-2 M arsenite on oxidation ofa-ketoglutarate by the cells grown in the absence

or presence of arsenate

Concn ofarsenate added Qo2 * Qo, with Inhibitionduring growth arsenite

M %0 286 12 96

10-2 272 228 17.510-3 266 227 14.5

* Expressed as ,umoles of oxygen consumed permg of cell N per 30 min at 30 C.

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arsenite. When cells were placed in medium con-taining 10-2 M arsenite at 30 C with shaking, arapid rise of arsenite resistance with a-keto-glutarate oxidation occurred prior to initiation ofgrowth (Fig. 5A). During this time, only a slightincrease of turbidity was observed. Full resistancewas reached within about 2 or 3 hr. Acquisition ofresistance to arsenite did not occur in the ar-senite-containing incubation medium which didnot support bacterial growth.When the resistant cells growing in the presence

of 10-2 M arsenite were resuspended in new me-dium without arsenite, the resistant character wasmaintained during about two generations (about2 hr) and then gradually disappeared (Fig. 5B).Mechanism of arsenite resistance in Pseudo-

rnonas. The results described above demonstratedthat resistance to arsenite in Pseudomonas was ac-quired through the process of physiological adap-tation. There are many possibilities concerningthe direct mechanism responsible for this resistantcharacter. Formation of alternative metabolicpathways insensitive to drugs, increased destruc-tion of drugs (Pollock, 1957), increase in amountsof enzyme inhibited by drugs (Mizushima andArima, 1960), increase in a content of a cofactorthat antagonizes drugs (Harington, 1959), anddecreased permeability of the cell to drugs (Izakiand Arima, 1963) have been postulated to beresponsible for drug resistance. These possiblemechanisms were examined in the experimentsdescribed below.

a-Ketoglutarate dehydrogenase activity in cell-freepreparations. Activities of the a-ketoglutaratedehydrogenase systems extracted from both sensi-tive and resistant cells required CoA for theiractivities and were both inhibited almost equallyby arsenite (Table 3). Also, a significant differencein their specific activity was not observed. Theseactivities were 135 units per mg of protein N inthe extracts of the sensitive cells and 111 in thoseof the resistant ones.

Contents of lipoic acid and sulfhydryl compoundsin the cells. The organisms grown with or without10-2 M arsenite were harvested, and appropriateamounts of the cells were hydrolyzed to determinecontents of lipoic acid and sulfhydryl compounds.Although considerable diversity of the values wasobserved, depending on the method used to pre-pare the cell hydrolysates, a significant increasein the contents of these compounds in the resistantcells could not be observed (Table 4).

Detoxication of arsenite. As reported by Mandel

A B

Cs

a50 r 50

0o

0 1 2 3 0 1 2 3 4 5

Hours Hours

FIG. 5. Appearance (A) and disappearance (B)of arsenite-resistant oxidation of a-ketoglutarateduring growth. Respiration resistant to 10-2 Marsenite is expressed in terms of 100 - % inhibitionby 10-2 M arsenite.

TABLE 3. Inhibition of ae-ketoglutarate dehydrogenaseactivity in cell-free preparations by arsenite

InhibitionArsenite concn added Sensitive Resistant

cells cells

M % %

1/3 X 104 27 302/3 X 10-4 52 59

TABLE 4. Contents of lipoic acid and total sulfhydrylcompounds in the cells

Lipoic acid Sulfhy-Strain ~~~~~~~~~drylStrain Alkaline Acid com-

hydrolysis hydrolysis pound*

,Ag pg pgSensitive cells. 1.8t 0.63 59Resistant cells ... 1.0 0.35 51

* Expressed as the amount of cysteine.t Results expressed as amount per mg of cell

N.

and Mayersak (1962), detoxication of arsenite byoxidation is one of the mechanisms of arseniteresistance. However, oxidation of arsenite couldnot be observed in the Pseudomonas cells bychemical estimation of arsenite.

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A B

100 _ 100 0O2 X 10-2

102

010 20 02

~. 50-50

2 x 10-2

0 00 ~~~10 20 0 10 20

Minutes Minutes

FIG. 6. Effect of cetyltrimethyl ammonium bromide-treatment on the arsenite-resistant oxidation of a-keto-glutarate. (A) Untreated resistant cells; (B) treated cells. The numbers indicate molarity of arsenite in theWarburg vessels.

Effect of cetyl-trimethylammonium bromide(CTAB) on the resistance to arsenite. There hasbeen considerable evidence suggesting that treat-ment of microorganisms with cationic detergents(for instance, CTAB) destroys the permeabilitybarrier of the cells to various metabolites (Bruem-mer and Wilson, 1961; Sato and Arima, in press).If this is true and arsenite resistance is caused bydecreased permeation of arsenite into the cells,then the treatment may decrease the degree ofarsenite resistance of Pseudomonas cells. This isthe case, as is illustrated in Fig. 6. Organismsgrown with 10-2 M arsenite were resuspended indistilled water, and CTAB was added to give afinal concentration of 100 ,g/ml. After incubationfor 10 to 20 min at 30 C with gentle shaking, thecells were recentrifuged and subjected to ma-nometry without washing. This treatment itselfdid not impair severely the oxidative activity of

a-ketoglutarate of the cell. Oxidation of a-keto-glutarate by the untreated organisms was notinhibited at all by arsenite at 2 X 10-2 M.However, remarkable inhibition was observedwhen the same concentration of arsenite wasadded to the organisms after treatment withCTAB.

DISCUSSIONThe process by which microorganisms acquire

resistance against various drugs may fall largelyin two different groups in nature: one involves ahereditary change in resistance to an inhibitor,and the other involves physiological changeswhich result in more or less temporary acquisitionof resistance. It can be concluded that the ar-senite resistance in the Pseudomonas cells de-scribed in this paper is a physiological phenom-enon induced by contact with the drug. The

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reasons are summarized as follows. (i) A veryshort lag time was observed with growth in thepresence of arsenite. It is too short to be ex-plained by the mutation theory (Fig. 1). (ii)Very rapid acquisiton of resistance was observedwith oxidation of a-ketoglutarate (Fig. 5A). (iii)Rapid disappearance of resistance was observedwhen the resistant organisms were transferred tomedium containing no arsenite (Fig. 5B). (iv)Induction of almost full resistance by contactwith an extremely low concentration of arsenitewas observed (Fig. 2); this excluded the possi-bility of selection of the mutant by the drug.

Inhibition sites of arsenate (As205) are knownto be distinct from those of arsenite (As203).However, mutual cross-resistance to arsenite andarsenate shown by the arsenate- and arsenite-grown cells indicates some common mechanismsin their resistance. Although the occurrence ofinterconversion between these two compounds byoxidation-reduction has been reported in thevarious bacteria (Turner, 1954; Turner andLegge, 1954), decrease of arsenite in culturesowing to oxidation to arsenate was not observedwith this organism.The intermediary formation of the dithiol form

of the lipoic acid coenzyme during the turnoverof the keto acid dehydrogenases renders theseenzymes particularly susceptible to inhibition byarsenite. As shown in Table 3, the properties ofa-ketoglutarate dehydrogenase in resistant cellswere not altered.An arsenite-resistance mechanism was reported

by Harington (1959) with blue tick. He observeda remarkable increase of the contents of sulf-hydryl compounds in an arsenic-resistant strainof blue tick larvae, and concluded that higherlevels of sulfhydryl compounds conferred uponthem a greater power to detoxicate given amountsof arsenicals. This mechanism was negated bythe results shown in Table 4.

Detoxication of arsenite by oxidation to ar-senate was reported by Mandel and Mayersak(1962) with Bacillus cereus. Though we could notprove the oxidation of arsenite by Pseudomonas,there were some possibilities that slow reactionoccurred in these organisms (Turner, 1954).However, it seems unlikely that the large amountsof arsenite (10-2 M) are detoxicated by the slowoxidation. The fact that the organisms grown inthe presence of arsenite acquired a resistantnature excluded detoxication as the mechanismsof resistance in this organism.

The effect of treatment with CTAB was re-cently confirmed with various microorganisms.Bruemmer et al. (1961) reported that treatmentof Azotobacter with CTAB unmasked its succin-oxidase activity. Sato et al. (in press) observedthat a similar treatment caused an appearanceof inhibitory effect of antimycin A on B. subtilis,which was not observed with the untreated or-ganisms. These results were attributed to destruc-tion of the permeability barrier of the cells forsuccinate and antimycin A, respectively. Thedata presented in Fig. 6 indicate decreased per-meation of arsenite in the resistant cells. Treat-ment with CTAB might facilitate penetration ofarsenite into the cells and cause it to have aninhibitory effect even on the resistant cells.

ACKNOWLEDGMENTThe authors wish to express their appreciation

to I. Tanabe, the Institute of Applied Microbi-ology, for his valuable advice in the taxonomicalstudies and K. Sakaguchi for his interest and en-couragement to this study. The authors acknowl-edge technical support from Fujisawa Pharma-ceutical Co. for bioassay of lipoic acid.

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